Diabetes mellitus is a serious metabolic disease that is defined by the presence of chronically elevated levels of blood glucose (hyperglycemia). This state of hyperglycemia is the result of a relative or absolute lack of activity of the peptide hormone, insulin. Insulin is produced and secreted by the β cells of the pancreas. Insulin is reported to promote glucose utilization, protein synthesis, and the formation and storage of carbohydrate energy as glycogen. Glucose is stored in the body as glycogen, a form of polymerized glucose, which may be converted back into glucose to meet metabolism requirements. Under normal conditions, insulin is secreted at both a basal rate and at enhanced rates following glucose stimulation, all to maintain metabolic homeostasis by the conversion of glucose into glycogen.
The term diabetes mellitus encompasses several different hyperglycemic states. These states include Type I (insulin-dependent diabetes mellitus or IDDM) and Type II (non-insulin dependent diabetes mellitus or NIDDM) diabetes. The hyperglycemia present in individuals with Type I diabetes is associated with deficient, reduced, or nonexistent levels of insulin which are insufficient to maintain blood glucose levels within the physiological range. Treatment of Type I diabetes involves administration of replacement doses of insulin, generally by a parenteral route. The hyperglycemia present in individuals with Type II diabetes is initially associated with normal or elevated levels of insulin; however, these individuals are unable to maintain metabolic homeostasis due to a state of insulin resistance in peripheral tissues and liver and, as the disease advances, due to a progressive deterioration of the pancreatic β cells which are responsible for the secretion of insulin. Thus, initial therapy of Type II diabetes may be based on diet and lifestyle changes augmented by therapy with oral hypoglycemic agents such as sulfonylureas. Insulin therapy is often required, however, especially in the latter states of the disease, in attempting to produce some control of hyperglycemia and minimize complications of the disease.
The structure and biology of amylin have previously been reviewed. See, for example, Young, Current Opinion in Endocrinology and Diabetes, 4:282-290 (1997); Gaeta and Rink, Med. Chem. Res., 3:483-490 (1994); and, Pittner et al., J. Cell. Biochem., 55S:19-28 (1994). Amylin is a 37 amino acid peptide hormone. It was isolated, purified and chemically characterized as the major component of amyloid deposits in the islets of pancreases of deceased human Type II diabetics (Cooper et al., Proc. Natl. Acad. Sci. USA, 84:8628-8632 (1987)). The amylin molecule has two important post-translational modifications: the C-terminus is amidated, i.e., the 37th residue is tyrosinamide, and the cysteines in positions 2 and 7 are cross-linked to form an intra-molecular N-terminal loop, both of which are essential for full biologic activity (Cooper et al., Proc. Natl. Acad. Sci. USA, 85:7763-7766 (1988)). Amylin is the subject of U.S. Pat. No. 5,367,052, issued Nov. 22, 1994.
In Type I diabetes and late stage Type II diabetes, amylin has been shown to be deficient and combined replacement with insulin has been proposed as a preferred treatment over insulin alone in all forms of insulin-dependent diabetes. The use of amylin and amylin agonists for the treatment of diabetes mellitus is the subject of U.S. Pat. No. 5,175,145, issued Dec. 29, 1992. Pharmaceutical compositions containing amylin and amylin plus insulin are described in U.S. Pat. No. 5,124,314, issued Jun. 23, 1992.
Excess amylin action has been said to mimic key features of the earlier stages of Type II diabetes and amylin blockade has been proposed as a novel therapeutic strategy. It has been disclosed in U.S. Pat. No. 5,266,561, issued Nov. 30, 1993, that amylin causes reduction in both basal and insulin-stimulated incorporation of labeled glucose into glycogen in skeletal muscle. The latter effect was also disclosed to be shared by calcitonin gene related peptide (CGRP) (see also Leighton and Cooper, Nature, 335:632-635 (1988)). Amylin and CGRP were approximately equipotent, showing marked activity at 1 to 10 nM. Amylin is also reported to reduce insulin-stimulated uptake of glucose into skeletal muscle and reduce glycogen content (Young et al., Amer. J. Physiol., 259:45746-1 (1990)). The treatment of Type II diabetes and insulin resistance with amylin antagonists is disclosed.
Amylin is primarily synthesized in pancreatic beta cells and is secreted in response to nutrient stimuli such as glucose and arginine. Studies with cloned beta-cell tumor lines (Moore et al., Biochem. Biophys. Res. Commun., 179(1) (1991)) and perfused rat pancreases (Ogawa et al., J. Clin. Invest., 85:973-976 (1990)) have shown that short pulses, 10 to 20 minutes, of nutrient secretagogues such as glucose and arginine, stimulate release of amylin as well as insulin. The molar amylin:insulin ratio of the secreted proteins varies between preparations from about 0.01 to 0.4, but appears not to vary much with acute stimuli in any one preparation. However, during prolonged stimulation by elevated glucose, the amylin:insulin ratio can progressively increase (Gedulin et al., Biochem. Biophys. Res. Commun., 180(1):782-789 (1991)). Thus, amylin and insulin are not always secreted in a constant ratio.
It has been discovered and reported that certain actions of amylin are similar to non-metabolic actions of CGRP and calcitonin; however, the metabolic actions of amylin discovered during investigations of this recently identified protein appear to reflect its primary biologic role. At least some of these metabolic actions are mimicked by CGRP, albeit at doses which are markedly vasodilatory (see, e.g., Leighton and Cooper, Nature, 335:632-635 (1988)); Molina et al., Diabetes, 39:260-265 (1990)).
The first discovered action of amylin was the reduction of insulin-stimulated incorporation of glucose into glycogen in rat skeletal muscle (Leighton and Cooper, Nature, 335:632-635 (1988)); the muscle, thus, became “insulin-resistant”. Subsequent work with rat soleus muscle ex-vivo and in vitro has indicated that amylin reduces glycogen synthase activity, promotes conversion of glycogen phosphorylase from the inactive b form to the active a form, promotes net loss of glycogen (in the presence or absence of insulin), increases glucose-6-phosphate levels, and can increase lactate output (see, e.g., International Patent Application No. PCT/US92/00185, published Jul. 23, 1992 (international Publication No. WO 92/11863). Amylin appears not to affect glucose transport per se (e.g., Pittner et al., FEBS Letts., 365(1):98-100 (1995)). Studies of amylin and insulin dose-response relations show that amylin acts as a noncompetitive or functional antagonist of insulin in skeletal muscle (Young et al., Am. J. Physiol., 263(2):E274-E281 (1992)). There is no evidence that amylin interferes with insulin binding to its receptors, or the subsequent activation of insulin receptor tyrosine kinase (Follett et at., Clinical Research, 39(1) :39A (1991)); Koopmans et al., Diabetologia, 34:218-224 (1991)).
It is believed that amylin acts through receptors present in plasma membranes. Studies of amylin and CGRP, and the effect of selective antagonists, have led to reports that amylin acts via its own receptor (Beaumont et al., Br. J. Pharmacol., 115(5):713-715 (1995); Wang et al., FEBS Letts., 219:195-198 (1991 b)), in contrast to the conclusion of other workers that amylin may act primarily at CGRP receptors (e.g., Chantry et al., Biochem. J., 277:139-143 (1991)); Zhu et al., Biochem. Biophys. Res. Commun., 177(2):771-776 (1991)). Amylin receptors and their use in methods for screening and assaying for amylin agonist and antagonist compounds are described in U.S. Pat. No. 5,264,372, issued Nov. 23, 1993.
While amylin has marked effects on hepatic fuel metabolism in vivo, there is no general agreement as to what amylin actions are seen in isolated hepatocytes or perfused liver. The available data do not support the idea that amylin promotes hepatic glycogenolysis, i.e., it does not act like glucagon (e.g., Stephens et al., Diabetes, 40:395-400 (1991); Gomez-Foix et al., Biochem J., 276:607-610 (1991)). It has been suggested that amylin may act on the liver to promote conversion of lactate to glycogen and to enhance the amount of glucose able to be liberated by glucagon. In this way, amylin could act as an anabolic partner to insulin in liver, in contrast to its catabolic action in muscle.
In fat cells, contrary to its action in muscle, amylin has no detectable actions on insulin-stimulated glucose uptake, incorporation of glucose into triglyceride, CO2 production (Cooper et al., Proc. Natl. Acad. Sci., 85:7763-7766 (1988)) epinephrine-stimulated lipolysis, or insulin-inhibition of lipolysis (Lupien and Young, “Diabetes Nutrition and Metabolism—Clinical and Experimental,” Vol. 6(1), pages 1318 (February 1993)). Amylin thus exerts tissue-specific effects, with direct action on skeletal muscle, marked indirect (via supply of substrate) and perhaps direct effects on liver, while adipocytes appear “blind” to the presence or absence of amylin.
It has also been reported that amylin can have marked effects on secretion of insulin. In the perfused pancreas (Silvestre et al., Reg. Pept., 31:23-31 (1990)), and in the intact rat (Young et at., Mol. Cell. Endocrinol., 84:R1-R5 (1992)), some experiments indicate that amylin inhibits insulin secretion. Other workers, however, have been unable to detect effects of amylin on isolated β-cells, on isolated islets, or in the whole animal (see Broderick et al., Biochem. Biophys. Res. Commun., 177:932-938 (1991) and references therein).
Amylin and amylin agonists have also been shown to suppress glucagon secretion. When influences that would otherwise affect glucagon secretion were controlled (plasma glucose, insulin and blood pressure), amylin reportedly suppressed the glucagon response to arginine in rats. Gedulin et al., Metabolism, 46:67-70 (1997). The amylin anaogue, pramlintide, has been reported to eliminate the post-prandial surge in glucagon concentration in subjects wth Type I diabetes. Fineman et al., Diabetes, 40:30A (1997). Pramlintide, and other amylin agonist analogues, are described and claimed in U.S. Pat. No. 5,686,411, issued Nov. 11, 1997. A glucagonostatic effect of amylin has not been demonstrated in the isolated perfused pancreas (Silvestre et al., Regul. Pept., 31:23-31 (1990), indicating that amylin may exert its glucagonostatic action via an extrapancreatic mechanism. The observation that suppression of glucagon secretion does not occur with the amylin analogue, pramlintide, in humans during insulin-induced hypoglycemia (Nyholm et al., J. Clin. Endocrin. Metab., 81:1083-1089 (1996); Kolterman et al., Diabetologia, 39:492-499 (1996)) further supports the idea that this effect is not directly on a cells but could be centrally mediated.
Amylin and amylin agonists potently inhibit gastric emptying in rats (Young et al., Diabetologia 38(6):642-648 (1995)), dogs (Brown et al., Diabetes 43(Suppl 1):172A (1994)) and humans (Macdonald et al., Diabetologia 38(Suppl 1):A32 (abstract 118) (1995)). Gastric emptying is reportedly accelerated in amylin-deficient Type I diabetic BB rats (Young et al., Diabetologia, supra; Nowak et al., J. Lab. Clin. Med., 123(1):110-6 (1994)) and in rats treated with the amylin antagonist, AC187 (Gedulin et al., Diabetologia, 38(Suppl 1):A244 (1995). The effect of amylin on gastric emptying appears to be physiological (operative at concentrations that normally circulate).
Non-metabolic actions of amylin include vasodilator effects which may be mediated by interaction with CGRP vascular receptors. Reported in vivo tests suggest that amylin is at least about 100 to 1000 times less potent than CGRP as a vasodilator (Brain et al., Eur. J. Pharmacol., 183:2221 (1990); Wang et al., FEBS Letts., 291:195-198 (1991)).
Injected into the brain, or administered peripherally, amylin has been reported to suppress food intake, e.g., Chance et al., Brain Res., 539:352-354 (1991)), an action shared with CGRP and calcitonin. The effective concentrations at the cells that mediate this action are not known. Amylin has also been reported to have effects both on isolated osteoclasts where it caused cell quiescence, and in vivo where it was reported to lower plasma calcium by up to 20% in rats, in rabbits, and in humans with Paget's disease (see, e.g., Zaidi et al., Trends in Endocrinal. and Metab., 4:255-259 (1993). From the available data, amylin seems to be 10 to 30 times less potent than human calcitonin for these actions. Interestingly, it was reported that amylin appeared to increase osteoclast cAMP production but not to increase cytosolic Ca2+, while calcitonin does both (Alam et al., Biochem. Biophys. Res. Commun., 179(1):134-139 (1991)). It was suggested, though not established, that amylin may act via a single receptor type whereas calcitonin may act via two receptors, one of which is common to amylin activity.
It has also been discovered that, surprisingly in view of its previously described renal vasodilator and other properties, amylin markedly increases plasma renin activity in intact rats when given subcutaneously in a manner that avoids any disturbance of blood pressure. This latter point is important because lowered blood pressure is a strong stimulus to renin release. Amylin antagonists, such as amylin receptor antagonists, including those selective for amylin receptors compared to CGRP and/or calcitonin receptors, can be used to block the amylin-evoked rise of plasma renin activity. The use of amylin antagonists to treat renin-related disorders is described in U.S. Pat. No. 5,376,638, issued Dec. 27, 1994.
In normal humans, fasting amylin levels from 1 to 10 pM and post-prandial or post-glucose levels of 5 to 20 pM have been reported (see, e.g., Koda et al., The Lancet, 339:1179-1180 (1992)). In obese, insulin-resistant individuals, post-food amylin levels can go higher, reaching up to about 50 pM. For comparison, the values for fasting and post-prandial insulin are 20 to 50 pM, and 100 to 300 pM respectively in healthy people, with perhaps 3- to 4-fold higher levels in insulin-resistant people. In Type I diabetes, where beta cells are destroyed, amylin levels are at or below the levels of detection and do not rise in response to glucose (Koda et al., The Lancet, 339:1179-1180 (1992)). In normal mice and rats, basal amylin levels have been reported from 30 to 100 pM, while values up to 600 pM have been measured in certain insulin-resistant, diabetic strains of rodents (e.g., Huang et al., Hypertension, 19:I-101-I-109 (1991)).
In mammals, calcitonin functions in the regulation of bone marrow turnover and calcium metabolism. Calcitonin, which is caused to be released from the thyroid by elevated serum calcium levels, produces actions on bone and other organs which tend to reduce serum calcium levels. Calcitonin inhibits osteoclast activity and reduces bone resorption, thereby reducing serum calcium levels. Calcitonin also alters calcium, phosphate and electrolyte excretion by the kidney, although the physiological significance of this is not reported. Calcitonin has been used clinically for treatment of disorders of calcium metabolism and pain, and its relationship to increased glucose levels in mammals has been the subject of varying reports. See, e.g., Azria et al., “Calcitonins—Physiological and Pharmacological Aspects,” pp. 24-25 (Springer-Verlag 1989). The use of calcitonins in the treatment of diabetes mellitus is described in U.S. Pat. No. 5,321,008 issued Jun. 14, 1994 and U.S. Pat. No. 5,508,260 issued Apr. 16, 1996.
Certain compounds reported to be calcitonin derivatives have been said to lower the calcium plasma level and to influence bone metabolism (U.S. Pat. No. 4,758,550 to Cardinaux et al.).